An easily performed fully automated and miniaturized procedure for the determination of antiknock agent ferrocene in gasoline is presented. The flow system was designed to focus on automation and miniaturization with minimal sample and reagent consumption by inexpensive instrumentation. As a result sample and reagent consumption was 0.4 mL. The procedure is based on on-line oxidation of ferrocene with benzoyl peroxide in gasoline-methanol mixture to color-forming ferrocenium-hydroxyl associate, followed by spectrophotometric determination in a stepwise injection system. The process of ferrocene oxidation to obtain color-forming ferrocenium-hydroxyl associate in homogeneous solution of gasoline sample in different organic solvents was studied. Under optimal experimental conditions (10 mM benzoyl peroxide, 0.1 mM HCl, oxidation time of 2 min) the linear detection range was found to be 1–100 mg L^− 1 in terms of Fe. The LOD, calculated from a blank test, based on 3σ, was found to be 0.3 mg L^− 1 in terms of Fe, with sample volume of 0.1 mL. The relative standard deviation for the determination of 1 mg Fe L^− 1 was 4.5% (n = 5). The sample throughput was 15 h^− 1. The proposed method was successfully applied for the determination of ferrocene in gasoline samples, and the analytical results agreed fairly well with the results obtained by reference UV-Vis and FAAS methods.